1. Field of the Invention
The present invention relates to a method for inducing mesenchymal stem cells from human pluripotent stem cells, mesenchymal stem cells produced by the method, and cell therapy products including the mesenchymal stem cells.
2. Discussion of the Background
Stem cells are cells which are capable of differentiating into a variety of cells constituting tissues of an organism, and generally refer to undifferentiated cells before differentiation, which can be obtained from respective tissues of an embryo, a fetus, and an adult body. The stem cells differentiating into specific cells by a differentiation stimulus (environment); allowing proliferation (expansion) thereof by producing the same cells as themselves through cell division (self-renewal), unlike cells of which cell division has been ceased due to completion of differentiation; and having plasticity in differentiation since they can differentiate into other cells under different environments or by different differentiation stimuli.
The stem cells may be classified into pluripotent, multipotent, and unipotent stem cells according to differentiation capability thereof. The pluripotent stem cells are pluripotent cells having totipotency to differentiate into all cells, and these include embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), etc. Adult stem cells may be examples of the multipotent and/or unipotent stem cells.
The embryonic stem S cells are formed from the inner cell mass of blastocyte in early embryogenesis; have totipotency to differentiate into all cells so that they can differentiate into any kind of tissue cells; can be cultured in an immortal and undifferentiated state; can be inherited to the next generation through preparation of germ cells, unlike the adult stem cells (Thomson et al., Science, 282; 1145-1147, 1998; Reubinoff et al., Nat, Biotechnol., 18; 399-404, 2000).
Human embryonic stem cells are prepared by isolating and culturing only the inner cell mass at the time of forming the a human embryo formation and, currently, the human embryonic stem cells prepared globally have been obtained from the frozen embryos remaining after sterilization operations. There have been various attempts to use pluripotent human embryonic stem cells that can differentiate into all cells as a cell therapy product; however, they have not yet completely overcome high barriers such as the risk of carcinogenesis and immunological rejection.
As one complement of these, induced pluripotent stem (iPS) cells have been reported recently. The iPS cells, which are included in the concept of the pluripotent stem cells, are cells obtained by de-differentiating adult cells of which differentiation is ended in several manners and thereby return them to embryonic-like state in an early stage of differentiation. So far, it has been reported that the de-differentiated cells exhibit almost the same characteristics as the embryonic stem cells, which are pluripotent stem cells, in view of gene expression and differentiation capability. These iPS cells can also use autologous cells and thereby exclude the risk of immunological rejection, however the risk of tumorigenesis still remain as a subject to be solved.
Recently, mesenchymal stem cells that have an immunoregulatory function and are free from the risk of tumorigenesis, have been presented as an alternative for solving such problems. The mesenchymal stem cells are multipotent cells which are capable of differentiating into adipocytes, osteocytes, chondrocytes, myocytes, neurocytes, cardiomyocytes, etc., and have been reported to have a function of regulating immune responses. The mesenchymal stem cells can be isolated and cultured from various tissues, but their capacity and cell surface markers are different from one another depending on the origins thereof. Therefore, it is not easy to clearly define the mesenchymal stem cells. However, the mesenchymal stem cells are generally defined by cells which can differentiate into osteocytes, chondrocytes and myocytes; have a spiral form; and express CD73(+), CD105(+), CD34(−), and CD45(−), which are basic cell surface markers.
Meanwhile, the minimal number about 1×109 of cells required in the fields of regenerative medicine and/or cell therapy needs to be satisfied, in order for the mesenchymal stem cells to be used as cell therapy products. However, the number of cells actually required is further increased, when considering experiments for setting conditions and standards. Therefore, at least 10 passages are needed for an in vitro experiment in order to supply such amount of cells from existing mesenchymal stem cells derived from various origins. In this case, the cells become aged and modified, and thus, they may not be adequate any more for the use as cell therapy products. Although the conditions and standards have been set by using these cells, some problems may occur that the cells might already become depleted before they are actually used in the therapy, so that the mesenchymal stem cells from others need to be used, and in that case, additional experiments need to be carried out due to the use of different cells.
The most ideal alternative to solve the above problems of the existing mesenchymal stem cell culturing system is to use human pluripotent stem cells to produce mesenchymal stem cells. However, so far, the induction of differentiation from human pluripotent stem cells into mesenchymal stem cells had required an induction procedure by a specific cytokine (e.g., BMP, bFGF), which costs much and needs control of concentration, or an induction procedure on xeno feeders (OP9 mouse cell lines) having the risk of xeno pathogen, and a sorting by a specific marker (e.g., CD73), thereafter.
Furthermore, as for the mesenchymal stem cells produced by these methods, it is difficult to maintain its fundamental state and production efficiency is not high. Moreover, human pluripotent stem cells having different genetic backgrounds have different physiological mechanisms, and thus cannot use the existing methods for inducing differentiation of mesenchymal stem cells, which were previously established in specific lines. Therefore, there was some difficulty that in order to induce mesenchymal stem cells from human pluripotent stem cells having different genetic origins, separate differentiation-inducing methods need to be developed and applied. For these reasons, the mesenchymal stem cells have limitations in being used as ideal cell therapy products in the fields of regenerative medicine and cell therapy.